Acta Biochim Biophys Sin, 2016, 48(3), 220–228 doi: 10.1093/abbs/gmv139 Advance Access Publication Date: 1 February 2016 Original Article
Original Article
Overexpression of miR-30a in lung adenocarcinoma A549 cell line inhibits migration and invasion via targeting EYA2 Yuncang Yuan1,†, Shangyong Zheng1,†, Qian Li1, Xudong Xiang2, Tangxin Gao1, Pengzhan Ran1, Lijuan Sun1, Qionglin Huang1, Fei Xie1, Jing Du1, and Chunjie Xiao1,* 1
School of Medicine, Yunnan University, Kunming 650091, China, and 2Department of Thoracic Surgery, Third Affiliated Hospital of Kunming Medical University, Kunming 650118, China †
These authors contributed equally to this work. *Correspondence address. Tel: +86-13888455098; Fax: +86-871-65034636; E-mail: [email protected]
Received 23 June 2015; Accepted 23 November 2015
Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs and closely related to the pathogenesis of cancers. Increasing evidence indicates that miR-30a plays a profound role during the development of cancers. However, the functions of miR-30a in non-small-cell lung cancer (NSCLC) are still ambiguous. Here we found that miR-30a was decreased in lung adenocarcinoma A549 cells and in tissue samples from 14 patients by qRT-PCR, and also found that overexpression of miR-30a in A549 cells inhibited migration and invasion but not cell proliferation and cell cycle progression by wound-healing assay, matrigel invasion assay, MTS-based cell proliferation assay, and flow cytometry-based cell cycle analysis, respectively. We further explored the potential mechanism of miR-30amediated gene regulation in lung adenocarcinoma cell lines. EYA2 is a predicted target of miR-30a, and it has been found that EYA2 expression is inhibited by miR-30a in breast cancer cells. We demonstrated that EYA2 is a direct target of miR-30a by using the dual-luciferase reporter assay in A549 cells and showed that EYA2 protein levels are inversely correlated with miR-30a expression in A549 and BEAS-2B cells. In addition, we also confirmed the rescue effects of EYA2 overexpression in A549 cells by cotransfection with EYA2 expression vector and miR-30a mimics. Taken together, our results demonstrate that overexpression of miR-30a in lung adenocarcinoma A549 cells can inhibit cell migration and invasion, which is partially attributed to the decrease of EYA2 expression. Our findings suggest that miR-30a may be used as a new potential target for the treatment of lung adenocarcinoma in the future. Key words: lung adenocarcinoma, miR-30a, migration, invasion, EYA2
Introduction Lung cancer is the main cause of cancer mortality in China, with an incidence of about 53.57 per 100,000 and a very high mortality in the registered areas in 2012 [1]. Non-small-cell lung cancer (NSCLC), including adenocarcinoma, squamous carcinoma, and large cell carcinoma, is the main histological type of the lung cancer
and accounts for nearly 80% of the lung cancer. Despite new drugs and therapeutic regimens, the overall five-year survival rate of NSCLC patients is still low at ∼15% at present. Effective therapeutic strategies are urgently needed for the NSCLC treatment. miRNAs are a class of conserved, 19–24 nucleotides long, noncoding small RNAs which act by fine-tuning gene expression through
© The Author 2016. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 220
miR-30a targets EYA2 in A549 cell a post-transcriptional mechanism. It is clear now that miRNAs perform their biological functions by guiding the RNA-induced silencing complex (RISC) toward 3′ UTR of target mRNAs [2]. miR-30a locates in human chromosome 6q13 and increasing evidence indicates that miR-30a can act as a tumor suppressor to inhibit the growth of various tumors including colorectal carcinoma [3–5], breast cancer [6], anaplastic thyroid cancer [7], Ewing’s tumor [8], clear cell renal cell carcinoma [9], gastric cancer [10], and nasopharyngeal carcinoma [10,11]. In addition, miR-30a also shows the feature of an oncogene. One study about glioma showed that miR-30a can promote tumor cell growth [12]. In a recent study, researchers also demonstrated the function of miR-30a as an oncogene in ovarian granulose cell tumor [13]. Together, these data indicate that the regulatory roles of miR-30a vary in different cancers. Currently, limited information concerning the biological functions of miR-30a in NSCLC has been reported. High-throughput screening results indicated that miR-30a expression was decreased in the NSCLC [14], but this may not be associated with the cell cycle, proliferation, xenograft formation, and chemosensitivity of A549 cells [15]. Considering the complexity of miRNAs’ regulation in cancers and the potential value of miRNAs in cancer treatment, further research is needed to address the functional consequence of abnormal miR-30a expression in the pathogenesis of NSCLC. EYA2 is a member of the EYA family proteins. As a novel class of protein tyrosine phosphatases (PTPs) [16], EYA2 has been reported to promote the proliferation, transformation, migration, and invasion of breast cancer [6,17]. The allosteric inhibitors of the EYA2 phosphatase had also been reported to inhibit EYA2-mediated cell migration in breast cancer [18]. Moreover, the abnormal expression of EYA2 was also found in other cancers such as cervical cancer [19], pancreatic adenocarcinoma [20], colorectal neoplasia [21], epithelial ovarian cancer [22], and particularly NSCLC [23]. A recent study demonstrated that EYA2 is a direct target of miR-30a in breast cancer [6]. Given that the mechanisms of miR-30a in NSCLC are still ambiguous, it is valuable to elucidate the relationship between miR-30a and EYA2 in NSCLC. In this study, the biological effects of miR-30a were studied in lung adenocarcinoma A549 cells. In addition, to provide more evidence for elucidating the mechanism of miR-30a in the pathogenesis of lung adenocarcinoma, EYA2 as a potential target of miR-30a was also explored in lung adenocarcinoma A549 cells. Our results indicated that overexpression of miR-30a in lung adenocarcinoma A549 cell line inhibited migration and invasion, which could partially be attributed to the decrease of EYA2 expression.
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Cell culture and transfection A549 lung carcinoma cells from the Tumor Hospital of Yunnan Province (Kunming, China) and BEAS-2B normal lung/bronchus epithelial cells from the Conservation Genetics CAS Kunming Cell Bank (Kunming, China) were cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, Rockford, USA) or Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS; Life Technologies, Grand Island, USA), 100 µg/ml streptomycin and 100 U/ml penicillin (Thermo Fisher Scientific). For functional analysis, siRNAs negative control (siRNAs NC), EYA2 siRNAs, inhibitors negative control (inhibitors NC), mimics negative control (mimics NC), miR-30a mimics, and miR-30a inhibitors (GenePharma, Shanghai, China) were transfected, respectively, into cells using HiPerFect Transfection Reagent (QIAGEN, Hilden, Germany). Plasmids and miR-30a mimics/mimics NC were cotransfected into cells using Attractene Transfection Reagent (QIAGEN). The transfection was done according to the manufacturer’s protocols.
Total RNA isolation and miRNA qRT-PCR analysis Total RNA was extracted from FFPE tissues using the miRNeasy FFPE Kit (QIAGEN) and from cell lines using Trizol reagent (TaKRa, Dalian, China) according to the manufacturer’s instructions. The stem-loop primer quantitative real-time polymerase chain reaction (qRT-PCR) method described by Chen et al. [24] was used to examine the expression level of endogenous miR-30a in lung tumor cells and tissues. U6 snRNA was chosen as an internal control. The primers used for PCR were as follows: miR-30a stem-loop primer, 5′-CAC AGCGGCTGTCGTTGACTGCGTGCTGCCGCTGTGCTTCCA-3′;
Materials and Methods Ethics statement The study was approved by the Medical Ethics Committee of the School of Medicine Yunnan University (Kunming, China). Human samples were used in accordance with the requirement of Medical Ethics Committee of the School of Medicine Yunnan University. All patients provided written informed consent.
Tissues Paired lung adenocarcinoma and adjacent normal tissues were obtained with diagnostic information from 14 patients who were diagnosed as lung adenocarcinoma from 2012 to 2013 at the No.1 School of Clinical Medicine, Kunming Medical University. All samples were formalin-fixed routinely and paraffin embedded (FFPE).
Figure 1. miR-30a is down-regulated in the lung adenocarcinoma cell line of A549 and adenocarcinoma tissues (A) The expression level of miR-30a in A549 and BEAS-2B cells was evaluated by qRT-PCR. U6 snRNA was used as an internal control. (B) The expression level of miR-30a in 14 pairs of adenocarcinoma tissues and their matched normal lung tissues were evaluated by qRT-PCR. U6 snRNA was used as an internal control. (C) The datasets GSE63805 searched from GEO were used for miR-30a expression analysis. **P < 0.01.
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Figure 2. Overexpression of miR-30a in A549 cells has no effect on the cell proliferation and cell cycle distribution. (A) For cell proliferation analysis, A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively. Twenty-four hours after transfection, cells were collected and seeded into a 96-well plate (1000 cells per well), and cultured for 120 h. The growth of the cells was analyzed by MTS assay, and all values were shown as the mean ± SE of triplicate measurements. (B) A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively, as indicated. Forty-eight hours later, 106 cells were collected for cell cycle analysis by PI staining and flow cytometer assay. (C) Western blot analysis of p27 kip1, cyclin D1, cyclin E1, and cyclin A1 in A549 cells after transfection with 20 nM mimics NC or miR-30a mimics. GAPDH was used as a loading control. All experiments were repeated at least three times with similar trends.
miR-30a forward primer, 5′-AGACCCGTGTAAACATCCTCG-3′; miR-30a reverse primer, 5′-GGCTGTCGTTGACTGCGTG-3′; U6 forward primer, 5′-CTCGCTTCGGCAGCACA-3′; and U6 reverse primer, 5′-AACGCTTCACGAATTTGCGT-3′. cDNA was synthesized by using the RT reagent kit (TaKRa), and PCR was performed by using SYBR Green real-time kit (TaKRa) in the ABI 7300 real-time PCR system (Life Technologies) with the following reaction conditions: initial denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 30 s, 60°C for 40 s, and then the dissociation curve was drawn.
Cell cycle analysis The A549 cells grown to 80%–90% confluence were detached by trypsinization at 48 h after transfection with 20 nM miR-30a mimics or mimics NC, fixed with 70% ethanol in PBS at −20°C overnight, washed with PBS and incubated at a density of 1–2 × 106 cells/ml with 50 µg/ml propidium iodide (PI) in PBS at room temperature in the dark for 10 min. DAPI fluorescence of cells was assayed by flow cytometry in the BD Accuri™ C6 Flow Cytometer (BD Biosciences, Franklin Lakes, USA).
Cell proliferation assay The A549 cells were seeded at 1000 cells per well into a 96-well plate (Corning Co, Corning, USA) and cultured at 37°C in 5% CO2. Twentyfour hours after transfection with 20 nM miR-30a mimics or mimics NC, cell proliferation was assessed every 24 h by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) assay using CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, USA) according to the manufacturer’s instructions. Briefly, MTS reagent solution was added to each well and incubated at 37°C in 5% CO2 for 2 h. Cell proliferation was assessed by measuring the absorbance at 490 nm with a SpectraMax 340PC384 Microplate Reader (Molecular Devices, Sunnyvale, USA). Triplicate wells were measured for cell viability in each treatment group.
Wound-healing assay The wound-healing assay mimics tumor cell migration in vivo. The A549 cells were seeded in a 6-well plate at a density of 0.5–1 × 106 cells/well, and after 24 h of growth they should reach ∼90% confluence as a monolayer. Then, cells were transfected with 20 nM miR-30a mimics or EYA2 siRNAs and their corresponding negative control. Then, the cells were cultured in DMEM with 2% FBS (lowserum culture media) for 12 h. The monolayer was scratched gently and slowly with a yellow pipette tip across the center of the well. After scratching, the detached cells were washed away with phosphatebuffered saline (PBS). The remaining cells were cultured for additional 48 h in fresh low-serum culture media to allow wound healing. At 0 and 48 h, images were captured on a photomicroscope (Olympus, Tokyo, Japan) at a magnification of ×200. The migration ability of the cells was assessed by counting the wound closure rate.
Matrigel invasion assay The A549 cells were harvested 24 h after transfection with 20 nM miR-30a mimics or EYA2 siRNAs and their corresponding negative
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Figure 3. Overexpression of miR-30a in A549 cells inhibited migration and invasion of the cells (A) The wound-healing assay of A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively. (B) The rate of wound closure ±SE was shown. The experiment was performed in triplicate with similar trend. (C) A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively, were seeded into the up chambers at 8000 cells per well. After culturing for 24 h, the invasive cells were stained by Giemsa. Representative fields of invasive cells on membrane are captured. (D) The average number of invasive cells per field from three independent experiments±SE was shown. **P < 0.01.
control, resuspended in FBS-free DMEM culture medium. Transwell chambers with 0.8-µm pore size filter (Corning) were coated with 30 µg of matrigel (BD Biosciences) and dried for 60 min at 37°C. The lower chamber was filled with 750 µl DMEM culture medium containing 10% FBS, and the upper chamber was seeded with 8000 cells from each treatment. Then, the chambers in 24-well plate were incubated at 37°C in 5% CO2 for 24 h. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 3.7% formaldehyde solution for 2 min, permeabilized by 100% methanol for 20 min, stained by Giemsa for 15 min at room temperature and washed with PBS. Cells remaining on the upper chamber (non-invaded) were scraped off gently with a cotton swab. The images of six random fields on the lower surfaces (with invasive cells) were captured by the photomicroscope at a magnification of ×200. The cells in each field were counted.
Dual-luciferase reporter assay To simplify our cloning strategy, the oligonucleotides in the 3′ UTR of the EYA2 mRNA ( position 2592-2702, NM_005244.4) which contains the seed sequence of the miR-30a were synthesized using a chemical-based oligonucleotide synthesis method (Sangon Biotech, Shanghai, China). As a negative control, oligonucleotides containing a mutated miR-30a targeting site were also designed. The mutants
were created by replacing the seed regions of the miR-30a targeting site with complementary bases. The restriction enzyme cleavage sites of XhoI and XbaI in the multiple cloning site (MCS) of the pGLO dual-luciferase miRNA target expression vector (Promega) were chosen as cloning sites. The oligonucleotides were inserted downstream of the firefly luciferase gene in pGLO vector, and the resulting plasmids were designated as pGLO-EYA2-wild-type and pGLO-EYA2-mutant, respectively. Renilla luciferase gene fused into pGLO vector was used as an internal control. The ligated product was transduced into DH5α (TaKRa). After 12 h of amplification at 37°C in a shaker (Thermo Fisher Scientific), the total plasmids were extracted from DH5α solution by the plasmid purification kit (QIAGEN) and sequenced (Sain Biological Corporation, Shanghai, China). For the luciferase reporter assay, A549 cells were co-transfected in a 24-well plate with 200 ng of the recombinant plasmids and 20 nM miR-30a mimics per well. Twenty-four hours after co-transfection, cell lysates were collected and luciferase activities were measured using a Dual-Luciferase Reporter System (Promega).
Western blot analysis Cells were lyzed on ice with lysis buffer containing protease inhibitors, phosphatase inhibitors, and phenylmethylsulfonyl fluoride. The lysates were clarified by centrifugation at 20,400 g for 10 min at
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in a 10 mM citrate buffer ( pH 6.0), and endogenous peroxidase activity was quenched by using 3% hydrogen peroxide in methanol for 10 min at room temperature. After being washed with PBS solution, sections were blocked with 10% normal goat serum for 10 min at room temperature. Then, the sections were incubated with a rabbit polyclonal anti-EYA2 antibody (Abcam) at a dilution of 1:1500 at 4°C overnight, followed by incubation with an anti-rabbit secondary antibody for 30 min at room temperature. Slides were then processed using 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin. Finally, the degree of immunoreactivity for EYA2 expression was evaluated semiquantitatively on the basis of staining intensity and the proportion of positive tumor cells. The staining intensity was graded as follows: 0 (no staining), 1 (light yellow), 2 (yellowish brown), and 3 (brown). The positive cells were graded according to the percentage of positive cells as follows: 0 (no positive tumor cells), 1 (80% positive tumor cells). The percentage of positive cells and the staining intensity were then multiplied to generate the immunoreactivity score.
Overexpression vector construction and rescue experiment
Figure 4. EYA2 gene is a direct downstream target of miR-30a in A549 cells (A) The EYA2-wild-type with putative binding site of miR-30a and the mutant generated by replacing the seed region of miR-30a with complementary bases were cloned into the pmirGLO vector, respectively. The restriction enzyme cutting sites of the XhoI and XbaI were chosen as the cloning sites. (B) For luciferase assay, A549 cells were transiently cotransfected with 200 ng luciferase vectors and 50 nM miR-30a mimics. Luciferase activities were measured 24 h after transfection. Firefly luciferase activities were normalized by the activity of renilla luciferase. The experiments were performed in triplicate with similar trend. **P < 0.01.
4°C, and the supernatants were collected. For each sample, 15 µg of total protein was electrophoresed on a 12% sodium dodecyl sulfate polyacrylamide gel electropheresis followed by an electrophoretic transfer to a polyvinyl difluoride membrane (Millipore, Massachusetts, USA). After being blocked for 60 min with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and 5% bovine serum albumin (BSA) at room temperature, the membranes were incubated with primary antibodies at 4°C overnight in 5% BSA, washed thrice with TBST, and then incubated with the corresponding horseradish peroxidase conjugate secondary antibody. The following primary antibodies were used: rabbit monoclonal anti-EYA2 antibody (Abcam, Cambridge, USA) at a dilution of 1:10,000; mouse monoclonal anti-GAPDH antibody (Abcam) at a dilution of 1:2000; rabbit monoclonal anti-p27 KIP1 antibody (Abcam) at a dilution of 1:2000; rabbit monoclonal anti-Cyclin E1 antibody (Abcam) at a dilution of 1:2000; rabbit polyclonal anti-Cyclin A1 antibody (BOSTER, Wuhan, China) at a dilution of 1:2000; and rabbit polyclonal anti-Cyclin D1 antibody (BOSTER) at a dilution of 1:2000. All bands were detected using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific).
Immunohistochemistry Lung adenocarcinoma FFPE tissues were cut into 4-µm thick sections. The sections were pretreated at room temperature for 1 h, then dewaxed in xylene, rehydrated in graded ethanol, and washed with PBS solution. Antigen retrieval was performed at 100°C for 20 min
The empty vector and pDoubleEx-EGFP-EYA2 expression vector were obtained from YRGene Bioresource Center (Changsha, China). To examine the transfection efficiency, enhanced green fluorescent protein (EGFP) expression in A549 cells was observed by an inverted fluorescence microscope (Olympus) at 48 h after transfection. Meanwhile, the cells were stained with Hoechst 33258 (Thermo Fisher Scientific). For a functional rescue experiment, A549 cells were cotransfected with p-EYA2 (2 µg) + miR-30a mimics (40 nM), p-EYA2 (2 µg) + mimics NC (40 nM), p-empty (2 µg) + miR-30a mimics (40 nM), and p-empty (2 µg) + mimics NC (40 nM), respectively, in a 6-well plate, and then the cells were used for the wound-healing assay and matrigel invasion assay as described previously.
Statistical analysis The 2-ΔΔCt method was used to analyze the relative changes in miR-30a expression from qRT-PCR experiments as described previously [25]. For the wound-healing assay, Image-Pro Plus 6.0 software (MediaCybernetics, Rockville, USA) was used to analyze the percentage of wound closure. ModFit LT 4.1 software (Verity, Topsham, USA) was used to analyze the cell cycle distribution of cells. All charts were generated by GraphPad Prism 5 software (GraphPad, La Jolla, USA). Differences in the numerical data between the cancer and adjacent normal tissues were analyzed by paired Student’s t-test. Other data were evaluated with one-way analysis of variance (ANOVA). All data were presented as the mean ± standard error (SE). A P value
Original Article
Overexpression of miR-30a in lung adenocarcinoma A549 cell line inhibits migration and invasion via targeting EYA2 Yuncang Yuan1,†, Shangyong Zheng1,†, Qian Li1, Xudong Xiang2, Tangxin Gao1, Pengzhan Ran1, Lijuan Sun1, Qionglin Huang1, Fei Xie1, Jing Du1, and Chunjie Xiao1,* 1
School of Medicine, Yunnan University, Kunming 650091, China, and 2Department of Thoracic Surgery, Third Affiliated Hospital of Kunming Medical University, Kunming 650118, China †
These authors contributed equally to this work. *Correspondence address. Tel: +86-13888455098; Fax: +86-871-65034636; E-mail: [email protected]
Received 23 June 2015; Accepted 23 November 2015
Abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs and closely related to the pathogenesis of cancers. Increasing evidence indicates that miR-30a plays a profound role during the development of cancers. However, the functions of miR-30a in non-small-cell lung cancer (NSCLC) are still ambiguous. Here we found that miR-30a was decreased in lung adenocarcinoma A549 cells and in tissue samples from 14 patients by qRT-PCR, and also found that overexpression of miR-30a in A549 cells inhibited migration and invasion but not cell proliferation and cell cycle progression by wound-healing assay, matrigel invasion assay, MTS-based cell proliferation assay, and flow cytometry-based cell cycle analysis, respectively. We further explored the potential mechanism of miR-30amediated gene regulation in lung adenocarcinoma cell lines. EYA2 is a predicted target of miR-30a, and it has been found that EYA2 expression is inhibited by miR-30a in breast cancer cells. We demonstrated that EYA2 is a direct target of miR-30a by using the dual-luciferase reporter assay in A549 cells and showed that EYA2 protein levels are inversely correlated with miR-30a expression in A549 and BEAS-2B cells. In addition, we also confirmed the rescue effects of EYA2 overexpression in A549 cells by cotransfection with EYA2 expression vector and miR-30a mimics. Taken together, our results demonstrate that overexpression of miR-30a in lung adenocarcinoma A549 cells can inhibit cell migration and invasion, which is partially attributed to the decrease of EYA2 expression. Our findings suggest that miR-30a may be used as a new potential target for the treatment of lung adenocarcinoma in the future. Key words: lung adenocarcinoma, miR-30a, migration, invasion, EYA2
Introduction Lung cancer is the main cause of cancer mortality in China, with an incidence of about 53.57 per 100,000 and a very high mortality in the registered areas in 2012 [1]. Non-small-cell lung cancer (NSCLC), including adenocarcinoma, squamous carcinoma, and large cell carcinoma, is the main histological type of the lung cancer
and accounts for nearly 80% of the lung cancer. Despite new drugs and therapeutic regimens, the overall five-year survival rate of NSCLC patients is still low at ∼15% at present. Effective therapeutic strategies are urgently needed for the NSCLC treatment. miRNAs are a class of conserved, 19–24 nucleotides long, noncoding small RNAs which act by fine-tuning gene expression through
© The Author 2016. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 220
miR-30a targets EYA2 in A549 cell a post-transcriptional mechanism. It is clear now that miRNAs perform their biological functions by guiding the RNA-induced silencing complex (RISC) toward 3′ UTR of target mRNAs [2]. miR-30a locates in human chromosome 6q13 and increasing evidence indicates that miR-30a can act as a tumor suppressor to inhibit the growth of various tumors including colorectal carcinoma [3–5], breast cancer [6], anaplastic thyroid cancer [7], Ewing’s tumor [8], clear cell renal cell carcinoma [9], gastric cancer [10], and nasopharyngeal carcinoma [10,11]. In addition, miR-30a also shows the feature of an oncogene. One study about glioma showed that miR-30a can promote tumor cell growth [12]. In a recent study, researchers also demonstrated the function of miR-30a as an oncogene in ovarian granulose cell tumor [13]. Together, these data indicate that the regulatory roles of miR-30a vary in different cancers. Currently, limited information concerning the biological functions of miR-30a in NSCLC has been reported. High-throughput screening results indicated that miR-30a expression was decreased in the NSCLC [14], but this may not be associated with the cell cycle, proliferation, xenograft formation, and chemosensitivity of A549 cells [15]. Considering the complexity of miRNAs’ regulation in cancers and the potential value of miRNAs in cancer treatment, further research is needed to address the functional consequence of abnormal miR-30a expression in the pathogenesis of NSCLC. EYA2 is a member of the EYA family proteins. As a novel class of protein tyrosine phosphatases (PTPs) [16], EYA2 has been reported to promote the proliferation, transformation, migration, and invasion of breast cancer [6,17]. The allosteric inhibitors of the EYA2 phosphatase had also been reported to inhibit EYA2-mediated cell migration in breast cancer [18]. Moreover, the abnormal expression of EYA2 was also found in other cancers such as cervical cancer [19], pancreatic adenocarcinoma [20], colorectal neoplasia [21], epithelial ovarian cancer [22], and particularly NSCLC [23]. A recent study demonstrated that EYA2 is a direct target of miR-30a in breast cancer [6]. Given that the mechanisms of miR-30a in NSCLC are still ambiguous, it is valuable to elucidate the relationship between miR-30a and EYA2 in NSCLC. In this study, the biological effects of miR-30a were studied in lung adenocarcinoma A549 cells. In addition, to provide more evidence for elucidating the mechanism of miR-30a in the pathogenesis of lung adenocarcinoma, EYA2 as a potential target of miR-30a was also explored in lung adenocarcinoma A549 cells. Our results indicated that overexpression of miR-30a in lung adenocarcinoma A549 cell line inhibited migration and invasion, which could partially be attributed to the decrease of EYA2 expression.
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Cell culture and transfection A549 lung carcinoma cells from the Tumor Hospital of Yunnan Province (Kunming, China) and BEAS-2B normal lung/bronchus epithelial cells from the Conservation Genetics CAS Kunming Cell Bank (Kunming, China) were cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, Rockford, USA) or Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) with 10% fetal bovine serum (FBS; Life Technologies, Grand Island, USA), 100 µg/ml streptomycin and 100 U/ml penicillin (Thermo Fisher Scientific). For functional analysis, siRNAs negative control (siRNAs NC), EYA2 siRNAs, inhibitors negative control (inhibitors NC), mimics negative control (mimics NC), miR-30a mimics, and miR-30a inhibitors (GenePharma, Shanghai, China) were transfected, respectively, into cells using HiPerFect Transfection Reagent (QIAGEN, Hilden, Germany). Plasmids and miR-30a mimics/mimics NC were cotransfected into cells using Attractene Transfection Reagent (QIAGEN). The transfection was done according to the manufacturer’s protocols.
Total RNA isolation and miRNA qRT-PCR analysis Total RNA was extracted from FFPE tissues using the miRNeasy FFPE Kit (QIAGEN) and from cell lines using Trizol reagent (TaKRa, Dalian, China) according to the manufacturer’s instructions. The stem-loop primer quantitative real-time polymerase chain reaction (qRT-PCR) method described by Chen et al. [24] was used to examine the expression level of endogenous miR-30a in lung tumor cells and tissues. U6 snRNA was chosen as an internal control. The primers used for PCR were as follows: miR-30a stem-loop primer, 5′-CAC AGCGGCTGTCGTTGACTGCGTGCTGCCGCTGTGCTTCCA-3′;
Materials and Methods Ethics statement The study was approved by the Medical Ethics Committee of the School of Medicine Yunnan University (Kunming, China). Human samples were used in accordance with the requirement of Medical Ethics Committee of the School of Medicine Yunnan University. All patients provided written informed consent.
Tissues Paired lung adenocarcinoma and adjacent normal tissues were obtained with diagnostic information from 14 patients who were diagnosed as lung adenocarcinoma from 2012 to 2013 at the No.1 School of Clinical Medicine, Kunming Medical University. All samples were formalin-fixed routinely and paraffin embedded (FFPE).
Figure 1. miR-30a is down-regulated in the lung adenocarcinoma cell line of A549 and adenocarcinoma tissues (A) The expression level of miR-30a in A549 and BEAS-2B cells was evaluated by qRT-PCR. U6 snRNA was used as an internal control. (B) The expression level of miR-30a in 14 pairs of adenocarcinoma tissues and their matched normal lung tissues were evaluated by qRT-PCR. U6 snRNA was used as an internal control. (C) The datasets GSE63805 searched from GEO were used for miR-30a expression analysis. **P < 0.01.
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Figure 2. Overexpression of miR-30a in A549 cells has no effect on the cell proliferation and cell cycle distribution. (A) For cell proliferation analysis, A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively. Twenty-four hours after transfection, cells were collected and seeded into a 96-well plate (1000 cells per well), and cultured for 120 h. The growth of the cells was analyzed by MTS assay, and all values were shown as the mean ± SE of triplicate measurements. (B) A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively, as indicated. Forty-eight hours later, 106 cells were collected for cell cycle analysis by PI staining and flow cytometer assay. (C) Western blot analysis of p27 kip1, cyclin D1, cyclin E1, and cyclin A1 in A549 cells after transfection with 20 nM mimics NC or miR-30a mimics. GAPDH was used as a loading control. All experiments were repeated at least three times with similar trends.
miR-30a forward primer, 5′-AGACCCGTGTAAACATCCTCG-3′; miR-30a reverse primer, 5′-GGCTGTCGTTGACTGCGTG-3′; U6 forward primer, 5′-CTCGCTTCGGCAGCACA-3′; and U6 reverse primer, 5′-AACGCTTCACGAATTTGCGT-3′. cDNA was synthesized by using the RT reagent kit (TaKRa), and PCR was performed by using SYBR Green real-time kit (TaKRa) in the ABI 7300 real-time PCR system (Life Technologies) with the following reaction conditions: initial denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 30 s, 60°C for 40 s, and then the dissociation curve was drawn.
Cell cycle analysis The A549 cells grown to 80%–90% confluence were detached by trypsinization at 48 h after transfection with 20 nM miR-30a mimics or mimics NC, fixed with 70% ethanol in PBS at −20°C overnight, washed with PBS and incubated at a density of 1–2 × 106 cells/ml with 50 µg/ml propidium iodide (PI) in PBS at room temperature in the dark for 10 min. DAPI fluorescence of cells was assayed by flow cytometry in the BD Accuri™ C6 Flow Cytometer (BD Biosciences, Franklin Lakes, USA).
Cell proliferation assay The A549 cells were seeded at 1000 cells per well into a 96-well plate (Corning Co, Corning, USA) and cultured at 37°C in 5% CO2. Twentyfour hours after transfection with 20 nM miR-30a mimics or mimics NC, cell proliferation was assessed every 24 h by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) assay using CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, USA) according to the manufacturer’s instructions. Briefly, MTS reagent solution was added to each well and incubated at 37°C in 5% CO2 for 2 h. Cell proliferation was assessed by measuring the absorbance at 490 nm with a SpectraMax 340PC384 Microplate Reader (Molecular Devices, Sunnyvale, USA). Triplicate wells were measured for cell viability in each treatment group.
Wound-healing assay The wound-healing assay mimics tumor cell migration in vivo. The A549 cells were seeded in a 6-well plate at a density of 0.5–1 × 106 cells/well, and after 24 h of growth they should reach ∼90% confluence as a monolayer. Then, cells were transfected with 20 nM miR-30a mimics or EYA2 siRNAs and their corresponding negative control. Then, the cells were cultured in DMEM with 2% FBS (lowserum culture media) for 12 h. The monolayer was scratched gently and slowly with a yellow pipette tip across the center of the well. After scratching, the detached cells were washed away with phosphatebuffered saline (PBS). The remaining cells were cultured for additional 48 h in fresh low-serum culture media to allow wound healing. At 0 and 48 h, images were captured on a photomicroscope (Olympus, Tokyo, Japan) at a magnification of ×200. The migration ability of the cells was assessed by counting the wound closure rate.
Matrigel invasion assay The A549 cells were harvested 24 h after transfection with 20 nM miR-30a mimics or EYA2 siRNAs and their corresponding negative
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Figure 3. Overexpression of miR-30a in A549 cells inhibited migration and invasion of the cells (A) The wound-healing assay of A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively. (B) The rate of wound closure ±SE was shown. The experiment was performed in triplicate with similar trend. (C) A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively, were seeded into the up chambers at 8000 cells per well. After culturing for 24 h, the invasive cells were stained by Giemsa. Representative fields of invasive cells on membrane are captured. (D) The average number of invasive cells per field from three independent experiments±SE was shown. **P < 0.01.
control, resuspended in FBS-free DMEM culture medium. Transwell chambers with 0.8-µm pore size filter (Corning) were coated with 30 µg of matrigel (BD Biosciences) and dried for 60 min at 37°C. The lower chamber was filled with 750 µl DMEM culture medium containing 10% FBS, and the upper chamber was seeded with 8000 cells from each treatment. Then, the chambers in 24-well plate were incubated at 37°C in 5% CO2 for 24 h. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 3.7% formaldehyde solution for 2 min, permeabilized by 100% methanol for 20 min, stained by Giemsa for 15 min at room temperature and washed with PBS. Cells remaining on the upper chamber (non-invaded) were scraped off gently with a cotton swab. The images of six random fields on the lower surfaces (with invasive cells) were captured by the photomicroscope at a magnification of ×200. The cells in each field were counted.
Dual-luciferase reporter assay To simplify our cloning strategy, the oligonucleotides in the 3′ UTR of the EYA2 mRNA ( position 2592-2702, NM_005244.4) which contains the seed sequence of the miR-30a were synthesized using a chemical-based oligonucleotide synthesis method (Sangon Biotech, Shanghai, China). As a negative control, oligonucleotides containing a mutated miR-30a targeting site were also designed. The mutants
were created by replacing the seed regions of the miR-30a targeting site with complementary bases. The restriction enzyme cleavage sites of XhoI and XbaI in the multiple cloning site (MCS) of the pGLO dual-luciferase miRNA target expression vector (Promega) were chosen as cloning sites. The oligonucleotides were inserted downstream of the firefly luciferase gene in pGLO vector, and the resulting plasmids were designated as pGLO-EYA2-wild-type and pGLO-EYA2-mutant, respectively. Renilla luciferase gene fused into pGLO vector was used as an internal control. The ligated product was transduced into DH5α (TaKRa). After 12 h of amplification at 37°C in a shaker (Thermo Fisher Scientific), the total plasmids were extracted from DH5α solution by the plasmid purification kit (QIAGEN) and sequenced (Sain Biological Corporation, Shanghai, China). For the luciferase reporter assay, A549 cells were co-transfected in a 24-well plate with 200 ng of the recombinant plasmids and 20 nM miR-30a mimics per well. Twenty-four hours after co-transfection, cell lysates were collected and luciferase activities were measured using a Dual-Luciferase Reporter System (Promega).
Western blot analysis Cells were lyzed on ice with lysis buffer containing protease inhibitors, phosphatase inhibitors, and phenylmethylsulfonyl fluoride. The lysates were clarified by centrifugation at 20,400 g for 10 min at
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in a 10 mM citrate buffer ( pH 6.0), and endogenous peroxidase activity was quenched by using 3% hydrogen peroxide in methanol for 10 min at room temperature. After being washed with PBS solution, sections were blocked with 10% normal goat serum for 10 min at room temperature. Then, the sections were incubated with a rabbit polyclonal anti-EYA2 antibody (Abcam) at a dilution of 1:1500 at 4°C overnight, followed by incubation with an anti-rabbit secondary antibody for 30 min at room temperature. Slides were then processed using 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin. Finally, the degree of immunoreactivity for EYA2 expression was evaluated semiquantitatively on the basis of staining intensity and the proportion of positive tumor cells. The staining intensity was graded as follows: 0 (no staining), 1 (light yellow), 2 (yellowish brown), and 3 (brown). The positive cells were graded according to the percentage of positive cells as follows: 0 (no positive tumor cells), 1 (80% positive tumor cells). The percentage of positive cells and the staining intensity were then multiplied to generate the immunoreactivity score.
Overexpression vector construction and rescue experiment
Figure 4. EYA2 gene is a direct downstream target of miR-30a in A549 cells (A) The EYA2-wild-type with putative binding site of miR-30a and the mutant generated by replacing the seed region of miR-30a with complementary bases were cloned into the pmirGLO vector, respectively. The restriction enzyme cutting sites of the XhoI and XbaI were chosen as the cloning sites. (B) For luciferase assay, A549 cells were transiently cotransfected with 200 ng luciferase vectors and 50 nM miR-30a mimics. Luciferase activities were measured 24 h after transfection. Firefly luciferase activities were normalized by the activity of renilla luciferase. The experiments were performed in triplicate with similar trend. **P < 0.01.
4°C, and the supernatants were collected. For each sample, 15 µg of total protein was electrophoresed on a 12% sodium dodecyl sulfate polyacrylamide gel electropheresis followed by an electrophoretic transfer to a polyvinyl difluoride membrane (Millipore, Massachusetts, USA). After being blocked for 60 min with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and 5% bovine serum albumin (BSA) at room temperature, the membranes were incubated with primary antibodies at 4°C overnight in 5% BSA, washed thrice with TBST, and then incubated with the corresponding horseradish peroxidase conjugate secondary antibody. The following primary antibodies were used: rabbit monoclonal anti-EYA2 antibody (Abcam, Cambridge, USA) at a dilution of 1:10,000; mouse monoclonal anti-GAPDH antibody (Abcam) at a dilution of 1:2000; rabbit monoclonal anti-p27 KIP1 antibody (Abcam) at a dilution of 1:2000; rabbit monoclonal anti-Cyclin E1 antibody (Abcam) at a dilution of 1:2000; rabbit polyclonal anti-Cyclin A1 antibody (BOSTER, Wuhan, China) at a dilution of 1:2000; and rabbit polyclonal anti-Cyclin D1 antibody (BOSTER) at a dilution of 1:2000. All bands were detected using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific).
Immunohistochemistry Lung adenocarcinoma FFPE tissues were cut into 4-µm thick sections. The sections were pretreated at room temperature for 1 h, then dewaxed in xylene, rehydrated in graded ethanol, and washed with PBS solution. Antigen retrieval was performed at 100°C for 20 min
The empty vector and pDoubleEx-EGFP-EYA2 expression vector were obtained from YRGene Bioresource Center (Changsha, China). To examine the transfection efficiency, enhanced green fluorescent protein (EGFP) expression in A549 cells was observed by an inverted fluorescence microscope (Olympus) at 48 h after transfection. Meanwhile, the cells were stained with Hoechst 33258 (Thermo Fisher Scientific). For a functional rescue experiment, A549 cells were cotransfected with p-EYA2 (2 µg) + miR-30a mimics (40 nM), p-EYA2 (2 µg) + mimics NC (40 nM), p-empty (2 µg) + miR-30a mimics (40 nM), and p-empty (2 µg) + mimics NC (40 nM), respectively, in a 6-well plate, and then the cells were used for the wound-healing assay and matrigel invasion assay as described previously.
Statistical analysis The 2-ΔΔCt method was used to analyze the relative changes in miR-30a expression from qRT-PCR experiments as described previously [25]. For the wound-healing assay, Image-Pro Plus 6.0 software (MediaCybernetics, Rockville, USA) was used to analyze the percentage of wound closure. ModFit LT 4.1 software (Verity, Topsham, USA) was used to analyze the cell cycle distribution of cells. All charts were generated by GraphPad Prism 5 software (GraphPad, La Jolla, USA). Differences in the numerical data between the cancer and adjacent normal tissues were analyzed by paired Student’s t-test. Other data were evaluated with one-way analysis of variance (ANOVA). All data were presented as the mean ± standard error (SE). A P value
